Fig 1: In LCWE-induced KD murine model, deficiency of platelet-miR-223 exacerbated the medial thickening, increased ICAM-1 expression with concomitant CD45+ inflammatory cells infiltration in the endothelium of abdominal aorta. LCWE or PBS was administrated i.p. for two weeks. The abdominal aorta tissues were collected, and continuous cross-sections were performed for H&E staining and immunofluorescence analysis. (A) Representative H & E-stained sections of PBS or LCWE injected mice were shown. n = 4, Scale bar: 50μm. (B) Quantification of the media layer areas of abdominal aorta in each group was shown. n = 4, Data are presented as mean ± SD, One-way ANOVA and Tukey’s multiple comparisons test. (C) Representative immunofluorescence images of sections from PBS or LCWE-injected mice. CD31 was stained as red, ICAM-1 as green, and the nucleus visualized as blue (DAPI). n = 4, Scale bars: 20μm. (D) Quantification of ICAM-1 expression in abdominal aorta tissues was shown (n = 4). Data are presented as mean ± SD, One-way ANOVA and Tukey’s multiple comparisons test. (E) Representative immunofluorescence images of sections from PBS or LCWE-injected mice. CD31 was stained as red, CD45 as green, and the nucleus visualized as blue (DAPI). n = 4, Scale bars: 20μm. (F) Quantification of CD45 expression in abdominal aorta tissues was shown (n = 4). Data are presented as mean ± SD, One-way ANOVA and Tukey’s multiple comparisons test. L, lumen; A, adventitia; Flox ctrl: miR-223 flox/flox mice.PF4-miR-223 KO, PF4-cre: miR-223 flox/flox mice. * P < 0.05, *** P < 0.001, **** P < 0.0001.
Fig 2: KD platelets were hyperactive and exhibit higher expression of miR-223. Plasma levels of PF4 (A) and β-TG (B) in HC and KD groups (HC: n = 24, KD: n = 24). Data are presented as median ± IQR, Mann Whitney test. (C) Surface expression of P-selectin (CD62P) in platelets isolated from HC and KD groups (HC: n = 45, KD: n = 45). Data are presented as mean ± SD, Unpaired t-test. (D) Relative expression of miR-223 in platelets isolated from HC and KD groups (HC: n=25, KD: n=25). Data are presented as median ± IQR, Mann Whitney test. ** P < 0.01, **** P < 0.0001. ns means no statistical difference.
Fig 3: The expression of PF4 and CXCR3 varied in different age groups. (A) Young and Old peripheral blood, as well as Cord Blood samples, were processed and evaluated using ELISA, qPCR and FACS. (B) ELISA quantification of PF4 in PRP lysate of Cord Blood (n = 15), Young (23.40 ± 2.13 years, n = 15) and Old (75.23 ± 4.19 years, n = 13) peripheral blood samples. (C) Quantitative real-time PCR analysis of PF4 expression in Cord Blood (n = 15), Young (n = 15) and Old (n = 13) peripheral blood mononuclear cells. (D) Flow cytometry analysis of CXCR3 expression in mononuclear cells from Cord Blood (n = 15), Young (n = 15) and Old (n = 13) peripheral blood. Data are presented as mean ± SD and analyzed using One-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001.
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